High-Resolution Epitope Mapping and Afﬁnity Binding Analysis Comparing a New Anti-Human LAG3 Rabbit Antibody Clone to the Commonly Used Mouse 17B4 Clone

: Lymphocyte activation gene 3 (LAG3) is a T cell inhibitory receptor that promotes tumor cell immune escape and is a potential target for cancer diagnostic and immunotherapeutic applica-tions. We used automated capillary electrophoresis (ACE), surface plasmon resonance (SPR), and immunohistochemistry (IHC) to compare the binding characteristics of a new anti-LAG3 rabbit antibody clone, SP464, with the thirty-year old and extensively used anti-LAG3 mouse 17B4 clone. The rabbit SP464 clone exhibited between 20 × to 30 × greater binding to LAG3 than did the mouse 17B4 clone. Using these tools, we precisely mapped the relative locations of the epitopes of these two antibodies. The SP464 and 17B4 minimal epitopes were localized to separate, but overlapping, sub-fragments within the amino-terminal ﬁfteen acids of the original thirty-mer peptide immunogen used to generate both antibodies. Application of this approach for quantifying the effects of alanine substitutions along the minimal SP464 epitope identiﬁed two amino acids essential for binding and four amino acids that likely contribute towards binding. Together, ACE, SPR, and IHC constitute a powerful orthologous approach for comparing antibody-binding characteristics and for ﬁne mapping of linear epitopes within short immunogens. Our results indicate that the rabbit clone SP464 may be useful for assessing LAG3 expression.

Introduction
The immunomodulatory protein, Lymphocyte activation gene 3 (LAG3) (CD223) was discovered over 30 years ago by Triebel et al. [1] as a novel member of the immunoglobulin (Ig) superfamily. LAG3 is a transmembrane protein with four extracellular Ig-like domains that show approximately 20% amino acid homology with CD4. Surface expression of the LAG3 receptor is induced on CD4+ and CD8+ T cells upon antigen stimulation, and functions as a negative regulator of T cell cytotoxicity. Recent studies have shown that the inhibitory function of LAG3 strongly correlates with the level of surface expression and subsequently, high LAG3, on tumor infiltrating lymphocytes, is associated with a poor prognosis [2,3]. Preclinical and early clinical trials have indicated that active LAG3 promotes tumor cell immune escape. Inhibition of LAG3 restores T cell activity, and is synergistic with inhibition of PD-1, making it a prime target for immunotherapy. The recently completed RELATIVITY-047 trial (Clinicaltrials.gov NCT0340922) demonstrated a significant improvement in median progression-free survival in patients with metastatic melanoma treated with a combination of anti-LAG3 BMS 986016 (Relatlimab), and anti-PD-1 (Nivolumab) compared to anti PD-1 alone [4]. This study led to FDA approval of a combination checkpoint inhibitor therapy for previously untreatable metastatic or unresectable melanomas. The emergence of LAG3 targeted therapies and co-development of immunotherapy with companion diagnostic (CDx) assays has established a need for new and well-characterized anti-LAG3 in vitro diagnostic reagents (IVDR) [5].
Immunohistochemistry (IHC) is a well-established technology for clinical diagnosis and CDx assays that is inexpensive and widely available; however, the utility of IHC is wholly dependent on reliable IVDR antibodies. The characterization and development of clinically useful antibodies faces a variety of persistent technical difficulties. In particular, identifying the precise location of an antibody binding site within a larger epitope is a critical step towards determining the specificity of an antibody for its target. A number of factors associated with identifying the actual binding sequence and characterizing binding with its cognate antibody are well documented [6]. Characterization studies include both physical (e.g., thermodynamic) and biological (e.g., site-directed mutation) analyses for characterizing antibody-protein interactions. Such studies require a number of tools, such as biosensor-based technologies, either surface plasmon resonance (SPR) [7,8] or bio-layer interferometry [9,10], ELISA [11], X-ray crystallography [12,13], nuclear magnetic resonance [14,15], mass spectroscopy [16,17], and bioinformatics aided by in silico technologies [18]. These techniques, when coupled with synthetic linear peptides, enables even more advancement in the area of fine epitope mapping techniques [19]. Efficiency improvements upon the 'Geysen pepscan' [20] and resulting multitude of techniques [21] have further expanded availability of these tools. However, fine epitope mapping has also identified some shortcomings of these tools when applied in a vacuum [14,22,23].
Currently, the most extensively used LAG3 specific antibody is the mouse anti-LAG3 monoclonal 17B4, which was developed thirty years ago [24]. Herein, we introduce a new rabbit-based clone, SP464, elicited from the same 30-mer immunogen as the 17B4 mouse clone and explore key binding differences between the clones. Additionally, we demonstrate the application of immunogen fragmentation and alanine-scanning mutagenesis to fine-map the minimum epitope sequences of both clones using the orthogonal technologies of Western blot, automated capillary electrophoresis (ACE), SPR, and IHC. The work presented here demonstrates the benefits of adapting this combinatorial approach as a standard method for development of an antibody as an IVD CDx assay.

Antibodies, Peptides and Proteins
The Roche Tissue Diagnostics VENTANA LAG3 rabbit (SP464) and mouse LAG3 (17B4) antibodies were from Spring Biosciences Corporation. Anti-FAPα antibody (SP325) and anti-CD8 alpha antibody (SP239) were obtained from Abcam. Peptides were synthesized by Anaspec, Freemont, CA. The lyophilized peptides were dissolved in dH2O, adjusted to a concentration of 2 mM, and stored at −80 • C until use. The recombinant LAG3 protein was from ABCAM.

Cell Lysates
Human lymphoma (HH, CRL-2105) cell line was from the ATCC. The human Hodgkin lymphoma (L-428, ACC 197) cell line was from the DSMZ. Human ataxia-telangiectasia mutated gene B lymphocyte cell line (AT, GM05126) was from the Coriell Institute. All cell lines were authenticated by the vendor, were maintained in a humidified incubator (37 • C, 5% CO 2 and 95% air), and used at less than 10 passages from origin. Cells were propagated in DMEM & RPMI 1640 medium, respectively, supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin at 37 • C with 5% CO 2 . Cells were cultured to the desired density (1 × 10 7 mL) and pelleted by centrifugation at 100× g at room temperature (RT) for 3 min. Pellets were washed with cold DPBS followed by 1× protease and phosphatase inhibitor solution in cold DPBS, and pelleted by centrifugation at 16,000× g at 4 • C for 20 min. Cell pellets were then lysed in RIPA lysis buffer supplemented with a protease and phosphatase inhibitor cocktail. Debris was removed by centrifugation at 15,000× g at 4 • C for 30 min, and the cleared lysate was stored at 4 • C.

Formalin Fixed Cell Line Blocks
Cells were propagated, harvested, and pelleted as described above. The cell pellets were suspended in phosphate buffered saline containing one percent low melt agarose. The agarose cell blocks were then formalin fixed and embedded in paraffin.

Tissue
Formalin fixed paraffin embedded (FFPE) tissues were commercially sourced and de-identified for use in research studies (US Biolabs).

Automated Capillary Electrophoresis (ACE) Reagents and Instrumentation
The WES anti-rabbit and anti-mouse Detection Modules and the 12-230 kDa Separation Module (8 × 25) were from ProteinSimple, San Jose, CA, USA. ACE assays were performed on a ProteinSimple WES instrument.

Surface Plasmon Resonance (SPR) Reagents and Instrumentation
SPR reagents and Series S CM5 sensor chips were obtained from Cytiva. SPR assays were performed on a BIAcore T200.

Western Blot and ACE Assays
Lysates were diluted in denaturing LDS sample load buffer and water to equal final volumes, denatured at 60 • C for 5 min, cooled and loaded onto a 4-12% polyacrylamide gradient gel followed by electrophoresis using NuPAGE MOPS Running Buffer for 1.5 h at 180-200 V. The gel was transferred with an iBLOT2 instrument to a 0.20 µm PVDF membrane for 7.5 min at 25 V. The membrane was then blocked for 0.5 h, washed with manufacturer supplied buffer, and then cut in half. One half was probed with rabbit SP464 antibody, and the other half with mouse 17B4 antibody. The membrane halves were incubated for 1 h at RT on a shaker in a 10 mL volume at a 1.0 µg/mL concentration of corresponding antibody diluted into WesternBreeze antibody diluent. The membranes were washed and incubated for 30 min at RT on a shaker with the corresponding anti-species antibody alkaline phosphatase conjugate. The membranes were washed, and antibody binding was detected with a chemiluminescent substrate according to the manufacturer's protocol and then imaged on a FluorChem HD2 Imager. A parallel lysate set used for the Western blot was prepared and processed on the WES instrument using the manufacturer's reagents and protocols as described below. As noted by the WES manufacturer, protein molecular weight size estimates can vary between standard Western blot and ACE methodologies.

ACE Antibody Titration Assays
For each assay run, HH cell lysate was mixed with 0.1× WES sample buffer reagent, and reconstituted WES 5× Fluorescent Mix reagent followed by denaturation at 95 • C for 3 min and then held on ice followed until use. Denatured sample was added to each of the designated wells of the separation module sampling plate. Dilutions of the stock rabbit SP464 and mouse 17B4 monoclonal antibodies were made to the desired concentrations using the WES detection module Antibody Dilution Buffer 2 reagent. Diluted antibody solutions were added to the designated wells of the separation module sampling plate. The remaining WES assay reagents were finally added to the plate and then processed in the WES instrument using the default running and detection parameters.

ACE Fragmented Immunogen and Alanine Substituted Peptide Inhibition Assays
HH cell lysate samples were prepared and used as above for antibody titration assays. Rabbit SP464 and mouse 17B4 antibodies were diluted to 4 µg/mL in the WES detection module Antibody Dilution Buffer 2 reagent. The antibody solutions were then diluted 1:1 (vol/vol) with varying concentrations of peptide, or diluent as a no peptide control, for 1 h at RT, and 10 µL of each antibody/peptide solution was added to the designated wells of the separation module sampling plate. Samples were run in duplicates or triplicates. Plates were then processed in the WES instrument using default running and detection parameters. The IC 50 value for each alanine-substituted peptide was determined using a non-linear fit of the log concentration of peptide inhibitor versus the combined peak areas of the soluble and full-length endogenous LAG3 bands using the four-parameter model of Prism GraphPad Prism with the bottom value constrained to zero.

SPR Epitope Binding Kinetics Assays
SPR epitope binding analysis was done as previously described [25,26]. Briefly, SPR kinetic analyses of peptide binding of both clones was performed on a BIAcore T200 using Series S CM5 sensor chips. The rabbit or mouse antibody capture surface was prepared with 50 ng/mL goat anti-species IgG Fc in 10 mM Acetate pH 5. Surface conditioning was performed with 50 nM of the species-specific whole molecule IgG for 120 s at 75 µL/min for five cycles. Rabbit surface regeneration was accomplished using a four step process: 15 s of 10 mM HEPES, 150 mM NaCl at 20 µL/min, 20 s of glycine pH 1.5 at 30 µL/min, and two injections of 30 s each with glycine pH 1.7 at 30 µL/min. Mouse surface regeneration was accomplished using a two-step process: two injections of glycine pH 1.7, one for 60 s at 30 µL/min and the other for 180 s at 20 µL/min. Kinetic measurements were performed at 25 • C in a solution of 10 mM HEPES, 150 mM NaCl, and 0.05% Tween. Antibody solution at 5 nM was captured for 120 s at 20 µL/min, and peptide interactions, visualized as binding curves, were measured for 400 s of association and 800 s of dissociation at 100 µL/min. Full immunogen peptide concentrations at 100, 33, 20, 11, and 3.7 nM were tested, with the 33 nM concentration measured in triplicate. Alanine substitution peptides were tested, without replicates, at 100 and 20 nM. Kinetic coefficients were calculated with BIAcore T200 Evaluation software using the 1:1 Langmuir binding model with global fit parameters and the bulk refractive index value held constant at zero. Curve fitting was done with least squares regression using Levenberg-Marquadt regression; half-life is calculated using the standard equation.

IHC Peptide Inhibition Assays
Cell line and tissue IHC samples were processed on a Roche Tissue Diagnostics Bench-Mark ULTRA automated staining platform using OptiView DAB detection chemistry (VMSI Cat# 760-700) with Hematoxylin II (790-2208) and Bluing (760-2037) counterstain. Bulk staining reagents were from Roche Tissue Diagnostics. The SP464/17B4 assay conditions consisted of 1 h of heat-based epitope retrieval on 4 µm thick sections of FFPE hepatocellular carcinoma specimens. Rabbit SP464 antibody was tested at a concentration of 49 nM (7.3 µg/mL) and mouse 17B4 antibody was tested at 263 nM (39.5 µg/mL). Primary antibody was pre-incubated with varying concentrations of peptide, for at least 60 min at 4 • C with mixing, before being incubated on-slide for 16 min at 37 • C. Each peptide was tested, in duplicate, at concentrations of 5000, 500, and 50 nM. Slides were scored by a qualified reader using the intensity of the DAB stain using a common 0 (no signal) to 3 (most intense) scale, and the percent of tumor tissue that is stained. LAG3 positive T cells typically display a punctate cytoplasmic staining pattern. Macrophages and plasmacytoid dendritic cells can also be stained with a lighter diffuse cytoplasmic pattern, however, for the purposes of this assay, that cell population is not included in the analysis. Digital images were captured on an Aperio imager at 40× magnification.

Chromogenic Multiplex IHC Assay
Fully automated multiplexed detection was performed on a Ventana Benchmark Ultra staining platform as described [27][28][29][30]. Detection was done using the chromogenic reagents 4-(4-dimethylaminophenylazo) benzensulfonyl (Dabsyl), carboxytetramethylrhodamine (TAMRA), and Cy5-based chromogens, available commercially in the DISCOVERY RUO Yellow kit (Cat. No. 760-239), DISCOVERY RUO Purple kit (Cat. No. 760-229), and DISCOVERY RUO Teal kit (Cat# 760-247), respectively. These kits also include the requisite enzyme-antibody conjugates. Serial tissue sections were stained with each marker and chromogen individually, and in a sequential multiplex assay. Briefly, slide mounted paraffin sections were deparaffinized and antigen retrieval was done using ULTRA Cell Conditioning 1 (950-224) incubation at 94 • C for 64 min. Staining of each biomarker was performed in sequential steps that included incubation with primary antibody at 37 • C, followed by secondary anti-species antibody conjugated to either peroxidase or alkaline phosphatase depending on whether the chromogen in sequence is a tyramide or quinone methide derivative. LAG3 (SP464) with Purple chromogen was stained first, CD8 (SP239) with Teal was stained second, and FAPα (SP325) with Yellow was stained third. Heat denaturation at 100 • C for 8 min in ULTRA Cell Conditioning 2 (950-223) was used to deactivate the antibody complex between staining cycles. Hematoxylin II (790-2208) and Bluing (760-2037) were used to counterstain. Slides were scanned at 20× magnification using a VENTANA DP200 imaging system.

Western Blot, IHC, and ACE Analysis of Rabbit SP464 and Mouse 17B4 Target Binding
To determine the specificity of the rabbit SP464 and mouse 17B4 antibodies, recombinant LAG3 and protein lysates from three human cell lines were analyzed by Western blot ( Figure 1A). Both antibodies detected a single band from the recombinant protein which migrates at about 53 kDa, with the SP464 signal appearing 2.5× more intense, as estimated by densitometry, compared to that of the 17B4 antibody. Two bands were observed using rabbit SP464 in the human lymphoma cell lines, one corresponding to soluble endogenous LAG3 (52 kDa) and a second higher intensity band corresponding to full length endogenous LAG3 (58 kDa). A similar banding pattern for the LAG3 recombinant protein and the three cell lines was also observed for the 17B4 antibody, however, the signal intensity for all bands was substantially less than observed for SP464. The same cell lysate preparations used for the Western blot assay were subjected to ACE analysis ( Figure 1B). The corresponding banding patterns and staining intensity for each antibody recapitulated those of the Western blot ( Figure 1D). FFPE cells from each cell line were stained by IHC with each antibody ( Figure 1C). The staining intensities paralleled those of both the Western blot and ACE analysis. For SP464, no staining was observed in the L428 negative control cell line, moderate staining was observed for the AT cell line, and intense staining was observed for cell line HH. A similar staining pattern was observed for 17B4, but the overall staining intensity was substantially reduced.

Binding Properties and Binding Kinetics of the Rabbit SP464 and Mouse 17B4 Antibodies
To examine the binding properties of the two anti-LAG3 clones, ACE was used to probe lysate from the highest expressing cell line, HH, with varying concentrations of each antibody (Figure 2A). The signal intensity increased with increasing concentrations of each antibody, however, the signal intensity of the mouse 17B4 antibody was substantially reduced compared to that of the rabbit SP464. A plot of the concentrations versus the signal for each antibody showed a typical hyperbolic binding curve ( Figure 2B). However, superposition of the curves at the same scale indicates that the maximal binding signal for SP464 is approximately 20× greater than for 17B4 ( Figure 2B).

Binding Properties and Binding Kinetics of the Rabbit SP464 and Mouse 17B4 Antibodies
To examine the binding properties of the two anti-LAG3 clones, ACE was used to probe lysate from the highest expressing cell line, HH, with varying concentrations of each antibody (Figure 2A). The signal intensity increased with increasing concentrations of each antibody, however, the signal intensity of the mouse 17B4 antibody was substantially reduced compared to that of the rabbit SP464. A plot of the concentrations versus the signal for each antibody showed a typical hyperbolic binding curve ( Figure 2B). However, superposition of the curves at the same scale indicates that the maximal binding signal for SP464 is approximately 20× greater than for 17B4 ( Figure 2B).
Both the mouse 17B4 and the rabbit SP464 antibodies were created using the same 30-mer immunogen peptide sequence, "GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY". The binding kinetics of each of the two antibodies for the immunogen were examined by SPR at 25 °C ( Figure 2C). The association rate constant (Ka) and dissociation rate constant (Kd) for SP464 was 2.8 × 10 5 M −1 s −1 and 1.2 × 10 −6 s −1 , respectively. The Ka and Kd for 17B4 was 5.4 × 10 6 M −1 s −1 and 6.6 × 10 −4 s −1 , respectively. The affinity (KD) for both SP464 and 17B4 was 4.1 × 10 −12 M and 1.2 × 10 −10 M, respectively. Despite a faster on-rate for the 17B4, the KD difference between the two clones is about 30×, arising from the dissociation rate Both the mouse 17B4 and the rabbit SP464 antibodies were created using the same 30-mer immunogen peptide sequence, "GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY". The binding kinetics of each of the two antibodies for the immunogen were examined by SPR at 25 • C ( Figure 2C). The association rate constant (Ka) and dissociation rate constant (Kd) for SP464 was 2.8 × 10 5 M −1 s −1 and 1.2 × 10 −6 s −1 , respectively. The Ka and Kd for 17B4 was 5.4 × 10 6 M −1 s −1 and 6.6 × 10 −4 s −1 , respectively. The affinity (KD) for both SP464 and 17B4 was 4.1 × 10 −12 M and 1.2 × 10 −10 M, respectively. Despite a faster on-rate for the 17B4, the KD difference between the two clones is about 30×, arising from the dissociation rate being approximately 500× faster for the mouse 17B4 than the rabbit SP464 antibody. Generally, run-to-run variance can have 3× range in values, while a 10× difference is a clear delimiter. This limit was used to define significance in the criticality of residues during the binding event in subsequent analyses.
being approximately 500× faster for the mouse 17B4 than the rabbit SP464 antibody. Generally, run-to-run variance can have 3× range in values, while a 10× difference is a clear delimiter. This limit was used to define significance in the criticality of residues during the binding event in subsequent analyses. Curves were generated from the quantitation of luminescent signal peak areas versus antibody concentration for the rabbit SP464 (black circles) and the mouse 17B4 (red triangles). Comparison of the clones using the same y-axis is shown on the right. (C) Antibody binding kinetics using SPR analysis was done for SP464 (left) and 17B4 (right) using increasing concentrations of the full-length (30-mer) immunogen. Different concentrations are displayed in colors, while the global curve fits are displayed with black lines. Both plots have the same axes for time scale and response units.

Mapping of Rabbit SP464 and Mouse 17B4 Epitopes on the 30-Mer Immunogen
To identify the specific epitope recognized by the two antibodies, three separate peptides spanning the N-terminus, the center, and the C-terminus of the 30-mer immunogen were used in an ACE-based peptide inhibition assay ( Figure 3). Decreasing concentrations of each synthetic peptide was incubated with each antibody and used to probe immobilized LAG3 from the HH cell lysate used above. The full-length immunogen eliminated antibody binding at concentrations greater than 50 nM for both 17B4 (Figure 3, Panel A) and SP464 (Figure 3, Panel B). The peptide consisting of the N-terminal 15 amino acids of the 30-mer immunogen was similarly effective in preventing antibody binding at concentrations of 5000 nM and 500 nM peptide. The peptide containing the central ten amino acids of the immunogen failed to inhibit either antibody at the same concentrations of peptide used for the N-terminal fragment, nor did a fragment containing the C-terminal 15 amino acids of the 30-mer immunogen inhibit either antibody. These data indicate that the epitope for both antibodies resides within the N-terminal 15 amino acids of the 30-mer immunogen. Further epitope mapping was done using three overlapping peptides from within this 15 amino acid sequence of the immunogen for both 17B4 (Figure 3, Panel C)

Mapping of Rabbit SP464 and Mouse 17B4 Epitopes on the 30-Mer Immunogen
To identify the specific epitope recognized by the two antibodies, three separate peptides spanning the N-terminus, the center, and the C-terminus of the 30-mer immunogen were used in an ACE-based peptide inhibition assay (Figure 3). Decreasing concentrations of each synthetic peptide was incubated with each antibody and used to probe immobilized LAG3 from the HH cell lysate used above. The full-length immunogen eliminated antibody binding at concentrations greater than 50 nM for both 17B4 (Figure 3, Panel A) and SP464 (Figure 3, Panel B). The peptide consisting of the N-terminal 15 amino acids of the 30-mer immunogen was similarly effective in preventing antibody binding at concentrations of 5000 nM and 500 nM peptide. The peptide containing the central ten amino acids of the immunogen failed to inhibit either antibody at the same concentrations of peptide used for the N-terminal fragment, nor did a fragment containing the C-terminal 15 amino acids of the 30-mer immunogen inhibit either antibody. These data indicate that the epitope for both antibodies resides within the N-terminal 15 amino acids of the 30-mer immunogen. Further epitope mapping was done using three overlapping peptides from within this 15 amino acid sequence of the immunogen for both 17B4 (Figure 3, Panel C) and SP464 (Figure 3, Panel D). Only the central ten amino acid peptide fragment inhibited binding of the mouse 17B4, while the peptide fragment consisting of the nine C-terminal amino acids inhibited binding of the rabbit, SP464. These data localize the epitope of the mouse 17B4 antibody to the nine amino acid sequence PAAAPGHPLA, and the epitope of the rabbit SP464 antibody resides in nine amino acid sequence PGHPLAPGP. es 2022, 11, x FOR PEER REVIEW and SP464 (Figure 3, Panel D). Only the central ten amino acid peptide fragment inhibited binding of the mouse 17B4, while the peptide fragment consisting of the nine C-terminal amino acids inhibited binding of the rabbit, SP464. These data localize the epitope of the mouse 17B4 antibody to the nine amino acid sequence PAAAPGHPLA, and the epitope of the rabbit SP464 antibody resides in nine amino acid sequence PGHPLAPGP.

Identification of Amino Acids in the SP464 Epitope Critical for Binding to Rabbit SP464
To determine the contribution of each amino acid residue to antibody recognition and fully map the epitope for the new antibody SP464, ACE, SPR, and IHC were used to examine the effect of alanine substitutions at each position in the SP464 epitope. The ability of varying concentrations of each substituted peptide (Figure 4) to inhibit SP464 binding to LAG3 after pre-incubation with SP464 antibody was analyzed by all three techniques. Comparison of ACE inhibition curves (Figure 4, column 2), SPR binding curves (Figure 4, column 3), and images of the IHC peptide inhibition (Figure 4, columns 4-6) are

Identification of Amino Acids in the SP464 Epitope Critical for Binding to Rabbit SP464
To determine the contribution of each amino acid residue to antibody recognition and fully map the epitope for the new antibody SP464, ACE, SPR, and IHC were used to examine the effect of alanine substitutions at each position in the SP464 epitope. The ability of varying concentrations of each substituted peptide (Figure 4) to inhibit SP464 binding to LAG3 after pre-incubation with SP464 antibody was analyzed by all three techniques. Comparison of ACE inhibition curves (Figure 4, column 2), SPR binding curves (Figure 4, column 3), and images of the IHC peptide inhibition (Figure 4, columns 4-6) are shown for each alanine substituted peptide (Figure 4, column 1). Quantitative metrics are summarized in Table 1. shown for each alanine substituted peptide (Figure 4, column 1). Quantitative metrics are summarized in Table 1.   The first column contains the name of the peptide in bold and the location of the alanine substitution in red. A81 is completely in bold as it the wildtype and has no substitution. Kinetic constants determined by SPR are provided describing association rate constant (Ka), dissociation rate constant (Kd), and affinity (KD). IHC score is provided per peptide inhibition concentration along with the percent of tumor that is positive in parentheses.
Alanine substitution at the eighty-first amino acid (A81) serves as a positive control for unaltered function since the alanine is naturally occurring. Binding inhibition as measured by ACE demonstrates an IC 50 of 11 nM and a Hill slope of −1.7. The SPR binding curve shows the characteristic SP464 shape, as shown in Figure 2C, with the straight-line signal during the dissociation phase, indicating that the peptide-antibody complex is very stable and not dissociating. Kinetically, the control has a 0.3 nM affinity based on an association of 1.9 × 10 5 M −1 s −1 and a dissociation of 5.5 × 10 −5 s −1 , resulting in a calculated half-life of 211 min. Stability of the complex was further demonstrated, as shown by the IHC images. In the absence of peptide, 50% of the tissue stains at the highest DAB intensity score of 3, as shown in Figure 4 and Table 1, for the L80 alanine substitution peptide. At near molar equivalent concentrations of peptide and antibody, the IHC signal is only modestly reduced in intensity, while higher concentrations of the peptide (5000 nM to 500 nM) are required to inhibit 50 nM of antibody binding to the target tissue.
Based on these values, cut-off distinctions between the wild type control peptide and the alanine substituted peptides were defined as follows: Critical residues are defined by the lack of an interaction (shown graphically) while KD values 10× greater than the A81 (control) peptide KD, 0.3 nM, are considered contributory. For IHC assays, tissue heterogeneity is a major factor of variability, and can contribute as much as 10% difference in percent coverage. Therefore, for this assay, an increase of 1-3% over baseline is considered not significant. Signal intensity changes of more than 0.5 from the highest score (3), are considered significant indicating partial inhibition, with a signal intensity of zero indicative of complete inhibition. Critical residues are those defined by a lack of inhibition at the highest peptide concentration. Full to partial inhibition at a peptide concentration of 500 nM is considered contributory. Two values derived from ACE analysis, the IC 50 and the Hill slope, were used in tandem to identify critical residues. An IC 50 greater than 10×, compared to the wildtype, is deemed critical. IC 50 values that are 2× or greater are considered contributory. Hill slope values 50% lower than the Hill slope of the wild type peptide, −1.7, are considered contributory. Experimental variance for ACE analysis has not been previously established, however small perturbations in binding characteristics can theoretically be used to delineate the minimum epitope.
All three techniques, ACE, SPR, and IHC demonstrate that amino acids H78 and L80 are critical residues, for SP464 epitope binding. As shown in Figure 4, ACE inhibition curves for alanine replacement of H78 and L80 demonstrated IC 50 values of 3600 nM and 6000 nM, respectively, a shift of more than 300× and 500×, when compared to the wildtype peptide. The SPR binding curves show no interaction between the antibody and peptide. IHC images show no inhibition of DAB signal at all concentrations; stain intensity and percent coverage are commensurate with peptide-free IHC staining.
Amino acids that contribute to SP464 binding but are not critical include G77, P79, P82 and G83. The ACE inhibition assay using the alanine substitution peptides G77, P79, and P82 produced IC 50 values that are more than 2× greater than the control peptide, but do not reach 10×. There is flattening of the H78, P79, and L80 peptide binding curves, resulting in a Hill slope value that approaches zero (greater than −1.7). The SPR binding curves of these peptides, and G83, show greater complex dissociation, compared to the control curves, with a dissociation 100× higher, representing faster dissociation. The resulting affinities are more than 10× greater than the control KD and the half-lives were reduced to times between 2 to 10 min. Similarly, the IHC images for the G77 substitution show DAB signal partially restored at both 500 and 50 nM concentrations, with lower signal intensity at 500 nM. The IHC images for P79, P82, and G83 substitutions also show partial inhibition of the DAB signal intensity at the highest concentration of peptide (5 µM). These data suggest that G77, P79, P82, and G83 contribute to the antibody affinity, but are not critical for epitope recognition.
The ACE metrics, IC 50 and Hill slope for P76, P84, H85, and P86, are nearly equivalent between these peptides and the control sequence. SPR binding curves closely resembled the control curves and the affinity for these peptide substitutions is less than 10× greater than the wild type KD. IHC images of these peptides used in the inhibition assay show DAB signal only at the lowest peptide concentration with a signal intensity representative of the peptide-free intensity. These data suggest that amino acids P76, P84, H85, and P86 may make minor contributions to antibody binding, however the nature of the interaction is unknown. The overall agreement between each of the techniques used for assessing the binding contributions of each of the amino acids within the SP464 epitope as determined by ACE, SPR, and IHC is good, suggesting overall that the minimal epitope likely consists of the sequence GHPLAPG. The comparative results are summarized in Table 2.

Application of SP464 in a Chromogenic Multiplex Assay
To investigate the potential of SP464 as an IVD reagent, FFPE sections from a non-small cell lung carcinoma (NSCLC) tissue were stained in a chromogenic multiplex IHC assay. Serial sections were stained using the anti-LAG3 clone SP464 (purple), anti-CD8 (SP239) (teal), and anti-Fibroblast activation protein, alpha (FAPα, SP325) (yellow) or sequentially with all three antibodies ( Figure 5). Anti-CD8, identifies T cells, and anti-FAPα, identifies tumor stroma. Activated T cells can be seen by co-expression of LAG3 (purple) primarily in a punctate cytoplasmic pattern within membrane stained CD8 positive T cells (teal) or as a blended blue-violet hue. Cells stained with LAG3 only (purple) are most likely tissue resident macrophages or plasmacytoid dendritic cells.
(teal) or as a blended blue-violet hue. Cells stained with LAG3 only (purple) are most likely tissue resident macrophages or plasmacytoid dendritic cells.

Discussion
The advent of precision medicine and the introduction of the drug-diagnostic co-development strategy has driven the need for reliable IVDRs that identify predictive biomarkers [31]. Biological reagents used for diagnostic and therapeutic purposes are often defined functionally and lack rigorous identification of the epitope target. Herein, we demonstrate that a combinatorial approach utilizing three powerful and orthogonal methods, ACE, SPR, and IHC can be used to not only elucidate the target epitope sequence, but can additionally characterize the specificity, affinity, and stability of an antibody-epitope interaction with commensurate accuracy.
Antibody characterization and target identification typically relies on Western blot analysis, in which the antibody is required to bind to a denatured epitope in the context of a full-length protein. Automated capillary electrophoresis, the ACE assay, is based on the Western blot principle; however, it utilizes micro volumes of sample separated in a gel coated capillary tube. We used a novel ACE approach to map the epitopes of the 17B4 and SP464 antibodies within the 30-mer immunogen peptide sequence using competitive Figure 5. Multiplex IHC of serial NSCLC tissue sections stained using traditional hematoxylin and eosin (H&E), anti-LAG3 clone SP464 (seen in purple), anti-CD8 (SP239) (seen in teal), and antifibroblast activation protein, alpha (FAPα, SP325) (seen in yellow). Chromogenic multiplex allows the visualization of all three antibodies on a single tissue section, as shown in the "Multiplex Result" panel.

Discussion
The advent of precision medicine and the introduction of the drug-diagnostic codevelopment strategy has driven the need for reliable IVDRs that identify predictive biomarkers [31]. Biological reagents used for diagnostic and therapeutic purposes are often defined functionally and lack rigorous identification of the epitope target. Herein, we demonstrate that a combinatorial approach utilizing three powerful and orthogonal methods, ACE, SPR, and IHC can be used to not only elucidate the target epitope sequence, but can additionally characterize the specificity, affinity, and stability of an antibody-epitope interaction with commensurate accuracy.
Antibody characterization and target identification typically relies on Western blot analysis, in which the antibody is required to bind to a denatured epitope in the context of a full-length protein. Automated capillary electrophoresis, the ACE assay, is based on the Western blot principle; however, it utilizes micro volumes of sample separated in a gel coated capillary tube. We used a novel ACE approach to map the epitopes of the 17B4 and SP464 antibodies within the 30-mer immunogen peptide sequence using competitive peptide inhibition assays with peptide sequence fragments derived from the immunogen. Further analysis identified both the critical and contributory residues for the SP464 antibody. Comparing across all three techniques, the substitution of the H78 and L80 residues with alanine results in complete loss of function, demonstrating that these amino acids are critical for the peptide-antibody complex. Furthermore, our approach revealed that the rabbit SP464 antibody epitope lies adjacent to and partially overlaps with that of the mouse 17B4 epitope.
To validate the sensitivity and epitope specificity identified by ACE, we used peptide binding kinetics by SPR, and functional assays by traditional IHC. The underlying technology of SPR enables the study of binding events with picomolar sensitivity, as peptide binding events on the chip surface alters the signal response. The IHC assay requires the antibody to recognize the protein in its native context within the cellular milieu and provides functional significance to the use of fine epitope mapping in IVDR development. For instance, comparisons of the SPR-based signal intensities show that the maximal binding signals were obtained for SP464, which exceeded those of 17B4 by approximately 20×, therefore suggesting that within an IVDR application, a wider range of LAG3 expression would be detected by SP464, than is possible with the mouse 17B4.
Immunotherapy with immune-checkpoint inhibitors has revolutionized cancer therapy. Companion and complementary diagnostic assays for PD-L1 have shown an association between tumor cell expression of PD-L1 and the efficacy of immune checkpoint inhibitors, however a subset of patients fail to respond in spite of high tumoral PD-L1 expression. One mechanism that may contribute to checkpoint inhibitor resistance is compromised T cell function due to inhibitory signals including LAG3 receptor activation. The recent FDA approval of the anti-PD1 and anti-LAG3 combination therapy highlights the need for greater precision in diagnostic assays to identify patients that may benefit from combination therapies. The RELATIVITY-047 clinical trial reported retrospective assessment of LAG3 expression in enrolled patients using a laboratory-developed test based on the 17B4 antibody [4,32]. Although the median progression-free survival was longer for patients with expression of 1% or greater, benefit was also seen when LAG3 was not detected, suggesting that an assay employing an anti-LAG3 monoclonal antibody with greater sensitivity and specificity could improve patient selection. Multiplexing assays utilizing highly sensitive and specific anti-LAG3 antibodies combined with additional biomarker specific antibodies, such as CD8, may prove useful for further assessing the relative biomarker distributions in various tumor types and thereby facilitate prognostic assessments [33,34].
Currently a number of mouse and rabbit anti-LAG3 monoclonal antibodies are commercially available, however, with the exception of the widely used mouse 17B4 clone, the epitopes and binding parameters of these other antibodies are either unknown or proprietary. In this study comparing the properties of the rabbit SP464 and mouse 17B4 clones, we have shown that a combination of ACE, SPR, and IHC constitutes an extremely powerful pre-clinical approach for epitope identification and for assessing and comparing the binding parameters of antibodies in development for clinical applications. This work establishes the technique and mathematical metrics in the novel usage of ACE for finely mapping a minimum epitope contained within a larger immunogen and identifying critical and contributory roles of specific amino acids within the epitope. The value of fine epitope mapping is the identification of the precise location of epitope boundaries and the amino acids that are critical for the antibody target epitope interaction. Knowledge of the precise sequence of a monoclonal antibody target epitope is critical for assessing the extent of target specificity. Peptide sequences in non-target proteins having either full or partial homology to the epitope of the target protein may detract from or even nullify the clinical utility of an antibody. This aids in the identification of cross-reactive sites in other proteins. Furthermore, by identifying critical contacts that are essential for binding, potentially crossreactive sequences, lacking one or more of the critical contact sites, can be excluded from a BLASTP protein sequence alignment analysis. Our approach enables the comparative characterization of antibody binding parameters, localizes the target epitope sequence within an immunogen, identifies amino acids that are critical and contributory to antibody binding, and facilitates bioinformatic identification of potential off-target sequences.


Antibacterial and Anti-Inflammatory Activities of Thymus vulgaris Essential Oil Nanoemulsion on Acne Vulgaris

Antibiotics are frequently used in acne treatment and their prolonged use has led to an emergence of resistance. This study aimed to investigate the use of natural antimicrobials as an alternative therapy. The antimicrobial and anti-inflammatory activities of five commonly used essential oils (EOs) (tea tree, clove, thyme, mentha and basil EOs), and their possible mechanisms of action against Cutibacterium acnes and Staphylococcus epidermidis, were explored. The effect of the most potent EO on membrane permeability was elucidated and its anti-inflammatory action, when formulated as nanoemulsion, was tested in an in vivo acne model. The in vitro studies showed that thyme EO had the most potent antimicrobial and antibiofilm activity, with phenolics and terpenoids as main antimicrobial constituents of EO. Thyme EO affected cell membrane permeability of both bacterial species, evident by the detection of the leakage of intracellular ions and membrane integrity by the leakage of nucleic acids. Morphological alteration in bacterial cells was confirmed by transmission electron microscopy. Thyme EO nanoemulsion led to the suppression of an inflammatory response in acne animal models along with a bacterial load decrease and positive histopathological changes. Collectively, thyme EO nanoemulsion showed potent antimicrobial and anti-inflammatory effects compared to the reference antibiotics, suggesting its effectiveness as a natural alternative in acne treatment.

Introduction
Acne vulgaris is one of the most common chronic inflammatory skin disorders. Almost 85% of individuals suffer from acne during their lifetime [1]. Acne causes significant morbidity and affects patients both physically and psychologically [2]. Acne is characterized by forming inflammatory and non-inflammatory lesions mainly on the face, neck, arms, upper trunk and back [3]. The pathogenesis of acne vulgaris is multifactorial, with contributing factors including a hormonally derived overproduction of sebum (seborrhea), follicular hyperkeratosis, changes in skin microbiota and immunological inflammatory responses [4]. These factors affect each other where seborrhea and increased keratinization of the follicular epithelium produce a favorable environment for the overgrowth of Cutibacterium acnes (previously known as Propionibacterium acnes) over Staphylococcus epidermidis and Staphylococcus aureus. Metabolites of these microorganisms induce follicular and perifollicular inflammation, especially due to the release of chemotactic substances [5].
Most anti-acne treatments include the use of antibiotics such as erythromycin and clindamycin [6]. However, these antibiotics can have adverse side effects on the skin's normal microbiota and hold the potential to cause the emergence of bacteria resistant to these antibiotics. To overcome the emergence of the resistance to conventional antibiotics, alternative natural antimicrobial agents have been tested for their potential effectiveness against acne. Essential oils (EOs) are one of the most important natural products derived from plants for their various biological properties and medicinal uses. EOs have been used around the world for centuries for various purposes. Ancient Egyptians have used aromatic oils as early as 4500 BC in cosmetics and ointments. EOs and their components are active against a variety of targets, particularly the membrane and cytoplasm, and in some cases, they completely change the morphology of the cells [7]. These oils are present as mixtures of various monoterpenes, alcohols, phenols, aldehydes and sulphur-containing compounds. Diverse mechanisms have been studied to elucidate the activity of an EO on bacterial cells. These mechanisms include degradation of the cell wall, damage to the cytoplasmic membrane, coagulation of the cytoplasm and leakage of cell contents due to increased permeability [8]. The hydrophobicity that is typical of EOs is responsible for the disruption of bacterial structures, leading to increased permeability due to an inability to separate the EOs from the bacterial cell membrane [9]. It is well established that C. acnes plays a pivotal role in the development of inflammatory skin diseases. Several studies have shown that infection with C. acnes involves an interaction with Toll-like receptors TLR-2 and TLR-4 on keratinocyte [10]. Activation of these pattern recognition receptors induces the release of inflammatory cytokines and chemokines, including tumor necrosis factor alpha (TNF-α) and interleukin (IL)-8, which mediate inflammatory responses in both keratinocytes and monocytes [11,12]. Both TNF-α and IL-8 reportedly exacerbate skin inflammation. Therefore, anti-acne treatment should have anti-inflammatory effects besides antimicrobial properties.
This study aimed to test the antimicrobial properties of five EOs commonly used in the Mediterranean region and their potency in treating acne vulgaris. Another goal of the current study was to investigate the possible mode of action of the most effective EO by elucidating its effect on the bacteria cell membrane and intracellular components. The study also aimed to develop a pharmaceutical formulation of the EO with the highest antimicrobial effect. Based on the in vitro antibacterial results of tested EOs against acnecausing bacteria, thyme EO was formulated as a nanoemulsion formula. Healing and anti-inflammatory properties of the developed nanoemulsion of thyme EO were tested in an animal model as a potential new formulation for acne treatment. To the best of our knowledge, this is the first study to explore the mechanism of action of thyme EO in healing acne, in addition to reporting the effective use of thyme EO as a nanoemulsion as a possible alternative anti-acne therapeutic agent.

Bacterial Strains and Their Maintenance
C. acnes standard strain ATCC 6919 was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA), while S. epidermidis DSM 28319 (equivalent to ATCC 35984) was obtained from the German Collection of Microorganisms (Braunschweig, Germany). C. acnes was cultured anaerobically on RCM agar (Oxoid Limited, Basingstoke, United Kingdom (UK) and incubated for 48 h at 37 • C under~5% CO 2 using an anaerobic jar and anaerobic atmosphere generation bags (Sigma-Aldrich, St. Louis, MO, USA). Isolated colonies of C. acnes were sub-cultured in an RCM broth for 48 h at 37 • C under anaerobic conditions. For S. epidermidis, the bacteria were cultured aerobically on brain heart infusion (BHI) agar (LAB M limited, Lancashire, UK) and incubated at 37 • C for 18 h. Isolated colonies of S. epidermidis were sub-cultured in BHI broth and incubated at 37 • C for 18 h aerobically. Glycerol stock of each bacterial strain were prepared using their appropriate media supplemented with 25% glycerol and stored in a −70 • C freezer (Thermo Fisher Scientific, Waltham, MA, USA).

Essential Oils (EOs)
The research-grade EOs used in this study are listed as Table 1 and classified according to the plant family names. The pharmaceutical-grade EOs were purchased from Haraz Microorganisms 2022, 10, 1874 3 of 24 herbal store (Cairo, Egypt). The composition of the most effective EOs was confirmed by gas chromatography-mass spectroscopy (GC-MS) analysis at the department of pharmacognosy, faculty of Pharmacy, Ain Shams University (Cairo, Egypt), as detailed in Section 2.6. Table 1. List of the EOs, their common names, family names and part of the plant used.

Animals
A total of 68 adult male BALB/c mice (25-35 g of weight) were used in the current study; mice were purchased from Theodor Bilharz Research Institute (Giza, Egypt). Mice were left to adapt to the study environment for at least two weeks before the start of each experiment. Mice were housed in a controlled environment at 25 ± 2 • C, 55 ± 5% humidity, and 12 h light/dark cycle. Animals were provided with a standard laboratory diet and water ad libitum. The research procedures were conducted in compliance with the principles and recommendations of the Guide for the Care and Use of Laboratory Animals Association, A.V.M (Institute of Laboratory Animal Resources (US) 1986). All the animal experiments were approved by the Research Ethics Committee (REC) of the Faculty of Pharmacy, Cairo University with the protocol identification code MIC2.3.1.

Screening of Antimicrobial Activity of the Selected EOs by Disc-Diffusion Method
As a preliminary step, antibacterial activities of all five EOs were determined using the agar disc-diffusion method according to Kirby-Bauer protocol (CLSI, 2006). EOs were diluted in sterile dimethyl sulphoxide (DMSO) analytical reagent (AR) grade, Loba Chemie (Mumbai, India), and stock solutions of each oil were prepared at a concentration range of 1.56-50%. DMSO and the prepared stock solutions were both sterilized by filtration through 0.2 µm syringe filters (Sigma-Aldrich, St. Louis, MO, USA). Bacterial suspensions of C. acnes and S. epidermidis were prepared, and optical density was adjusted to 0.5 at OD 600 nm. Bacterial suspensions were spread on RCM and BHI agar plates for testing EOs against C. acnes and S. epidermidis, respectively. Sterile filter-paper discs (6 mm in diameter) were loaded with 10 µL of DMSO or EOs at different concentrations (1.56% to 50%) and placed on the surface of the agar, then incubated under appropriate conditions detailed for each bacterial strain, as mentioned in Section 2.1. Standard antibiotics, clindamycin (2 µg/disc) and erythromycin (15 µg/disc), were used as positive controls, while DMSO was used as a negative control. The antibacterial activity of each EO at each concentration was evaluated by measuring the diameter of the zone of inhibition expressed in millimeters (mm). Each assay was performed in triplicate and the whole experiment was repeated at least three times.

Determination of MIC and MBC of the Screened EOs against C. acnes and S. epidermidis
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EOs were determined using the broth microdilution method in 96-well Ushaped bottom microtiter plates according to the Clinical Laboratory Standards Institute (CLSI)-2011. The EOs were dissolved in sterilized DMSO at a final concentration of 50% (v/v), then two-fold serial dilutions were performed from 25% to 1.03% (v/v) for each EO. Bacterial suspensions of C. acnes and S. epidermidis were prepared in RCM and BHI broth, respectively. The OD 600 nm was adjusted to 0.5, then 25 µL of each bacterial suspension, 25 µL of sterile RCM/BHI broth and 50 µL of each concentration of each EO Changes in the bacterial density were measured using a microplate reader at 620 nm. The MIC of the EO was determined as the lowest concentration of EO, at which no detectable bacterial density was observed at OD 600nm [13]. Sterile broth and DMSO were used as negative controls. For MBC determination, 10 µL of the mixtures of each bacterial suspension and EOs at different concentrations were inoculated on RCM and BHI agar for C. acnes and S. epidermidis, respectively. MBC was determined as the lowest concentration of the EO, at which the incubated bacterial strain showed no detectable colonies on its respective agar medium plates. The assays were performed in triplicate and repeated as three independent experiments.

Determination of the Chemical Composition of the Most Effective EOs by Gas Chromatography-Mass Spectroscopy
The chemical components of the most effective EOs were analyzed at the Faculty of Pharmacy Ain Shams University, using GC-MS on a Shimadzu GCMS-QP2010 (Koyoto, Japan) equipped with Rtx-5MS fused bonded column (30 m × 0.25 mm i.d. × 0.25 µm film thickness) (Restek, CA, USA) equipped with a split-splitless injector. The following operating conditions were used where the initial column temperature was kept at 45 • C for 2 min and programmed to 300 • C at a rate of 5 • C/min, and kept constant at 300 • C for 5 min. The injector temperature was 250 • C. The helium carrier gas flow rate was 1.41 mL/min. All the mass spectra were recorded applying the following conditions: filament emission current was 60 mA; ionization voltage at 70 eV; ion source at 200 • C. Diluted samples (1% v/v) were injected with split mode (split ratio 1: 15). The resultant peaks were identified using AMDIS software by comparing their retention indices (RI), retention times and mass spectra to the authentic samples on the NIST, Wiley library database (>90% match).

Determination of Minimum Biofilm Inhibitory Concentration
The minimum biofilm inhibitory concentration (MBIC) for EOs was determined against S. epidermidis using the microtiter plate method [14]. In each well, 100 µL of S. epidermidis in BHI-1% glucose (w/v) with OD 600 nm adjusted to 0.5 was mixed with 100 µL of EO at different concentrations 25-0.78% (v/v) then incubated at 37 • C for 24 h. Following incubation, the contents of the wells were removed and gently rinsed twice with 250 µL of phosphate-buffered saline (PBS). The plate was left to air-dry overnight. The formed biofilm was then stained with 0.5% (w/v) crystal violet for 30 min at room temperature. The excess crystal violet was washed with 300 µL distilled water per well. The washing step was repeated three times then the plate was air-dried overnight. To measure the stained biofilm, crystal violet was solubilized using ethanol 95% (v/v) and the color intensity was measured at 595 nm using a microplate reader. Sterile BHI broth was used as a negative control and S. epidermidis cell culture without EOs was used as a positive control. MBIC was determined as the concentration of EO, at which the OD 595 nm was equal to that of the negative control [15]. Experiments for each EO concentration were performed in triplicate and the assay was repeated three independent times.

Determination of the Minimum Biofilm Eradication Concentration
The minimum biofilm eradication concentration (MBEC), the lowest concentration of EO necessary to completely eradicate preformed biofilm for EOs, was determined against S. epidermidis using the microtiter plate method [14]. In each well, 200 µL of S. epidermidis suspension in BHI-1% glucose (w/v) with OD 600 nm adjusted to 0.5 were incubated for 24 h at 37 • C to allow biofilm formation. Following incubation, the bacterial suspension was discarded, and the wells were rinsed by flooding with sterile PBS twice then left to air-dry overnight. A total of 200 µL of each EO concentration was added to the wells and incubated at 37 • C for 24 h. After incubation, the contents of the wells were removed and gently rinsed twice with 250 µL of PBS. The plate was left to air-dry overnight and the remaining biofilm was stained with 0.5% (w/v) crystal violet for 30 min at room temperature. The excess crystal violet was washed with 300 µL of distilled water per well. The washing step was repeated three times then the plate was air-dried overnight. To determine the MBEC, crystal violet was solubilized using ethanol 95% (v/v) and the color intensity was measured at 595 nm using a microplate reader. The controls were the untreated biofilm and the sterile BHI-1% glucose broth (w/v).

Determination of Time-Kill Kinetics of Selected EO
The time-kill kinetics of thyme EO against C. acnes and S. epidermidis were performed according to a previously established protocol [16,17]. Bacterial suspensions at an initial inoculum of 10 8 CFU/mL were used. The time-kill kinetics of the selected EO were assayed at concentrations of 0.053 mg/mL, 0.106 mg/mL, and 0.212 mg/mL, equivalent to 1, 2 and 4 MIC. Different concentrations of selected EO were incubated with the bacterial cultures and killing capacity at 0, 1, 2, 4, 8 and 12 h, and were assessed using broth micro-dilution method. At each time point, 10 µL of the assay solution was withdrawn to make ten-fold serial dilutions and subjected to viable colony counts on BHI and RCM agar plates for the respective bacteria. Plates were incubated at 37 • C for 24 h under aerobic conditions and 48 h under anaerobic conditions for each respective bacterial strain. DMSO, BHI broth and RCM broth were used as negative controls. Each concentration of EO was assayed as triplicate and the entire assay was repeated two independent times.

Assessment of Possible Mechanisms of Action of Selected EO against Acne Associated Microbes
The most effective selected EO was assayed for its possible anti-acne mechanism of action using multiple assays, including induced morphological alterations in the tested bacteria, loss of membrane integrity and leakage of intracellular components. C. acnes and S. epidermidis suspensions were incubated overnight in appropriate broth media supplemented with 1% glucose at 37 • C. Following incubation, bacterial density was adjusted to 0.5 at OD 600 nm . A clear sterile cover slide was then added to each well of a six-well plate. In one set of wells, 250 µL of the selected EO at its MBIC concentration was added to an equal volume of C. acnes or S. epidermidis and incubated at 37 • C for 48 h and 24 h, respectively. In another set of wells, 250 µL of DMSO was added to an equal volume of C. acnes or S. epidermidis and served as the negative control. Following incubation, cover slides were gently washed with saline and prepared for SEM photography using JEOL model JSM-5200F electron microscope 25 kV, with a resolution of 5.5 nm and magnification power of 15× to 200,000×, JEOL Ltd. (Tokyo, Japan) at the Faculty of Agriculture, Cairo University, Cairo, Egypt.

Visualization of the Effect of the Selected EO on Bacterial Cells by Transmission Electron Microscopy
Cellular morphological alterations of bacteria treated with the selected EO was observed by transmission electron microscopy (TEM). Bacterial suspensions of C. acnes and S. epidermidis were prepared, their OD 600 nm was adjusted to 1 then the MBC of the selected EO was added to each suspension and incubated for 6 h. The bacterial suspensions were then centrifuged and washed twice using a PBS and treated with 2.5% glutaraldehyde overnight. The samples were prepared according to a previously published protocol [18] and observed by TEM photography using JEOL model JEM-1400Flash electron microscope 120 kV, with a resolution of 0.2 nm and magnification power of 10× to 1,200,000×, JEOL Ltd. (Tokyo, Japan) at the Faculty of Agriculture, Cairo University, Cairo, Egypt.

The Effect of the Selected EO on Bacterial Membrane Integrity The Effect of the Selected EO on Potassium Ions Permeability
Bacterial suspensions of C. acnes and S. epidermidis were prepared according to a previously published protocol [19]. Isolated colonies (5-7 colonies) of C. acnes and S. epidermidis were inoculated in RCM and BHI broth, respectively, then incubated at 37 • C for 48 h and 24 h, respectively. Following incubation, bacterial cells were centrifugated at 10,000 rpm for 10 min and washed twice with PBS and resuspended in saline. The optical density was adjusted at 600 nm to 1. The bacterial cells were then treated with the MIC of the selected EO or DMSO (negative control) for 6 h and centrifuged at 10,000 rpm for 10 min. After centrifugation, the amounts of potassium (K + ) were measured in the supernatant using the ICP spectrometry technique [20] at the Faculty of Agriculture, Ain Shams University, Cairo, Egypt.
The Effect of the Selected EO on 260 nm Absorbing Material (Nucleic Acids) The loss of nucleic acids through increased membrane permeability was assessed according to a previously published protocol [21] with some modifications. Bacterial suspensions of C. acnes and S. epidermidis were prepared. Isolated colonies (5-7 colonies) of C. acnes and S. epidermidis in RCM and BHI broth were incubated at 37 • C for 48 h and 24 h, respectively. After incubation, the bacterial suspensions were centrifuged at 10,000 rpm for 5 min, washed twice with PBS then resuspended in saline to adjust the optical density at 600 to 1. Resuspended bacteria in saline were treated with either DMSO or clindamycin (Dalacin C 600 mg/4 mL ampule, Pfizer) as negative controls, whereas vancomycin (Vancomycin 500 mg/10 mL ampoule, Mylan) was used as a positive control. The selected EO was tested at a final concentration equivalent to its MIC. Samples from bacterial cultures under each treatment were withdrawn after 30, 60 and 120 min, and were filtered through a 0.2-µm filter. The OD 260 nm of each test filtrate was measured using UV-Visible Spectro nanophotometer (IMPLEN GmbH, Munich, Germany).

The Effect of the Selected EO on Leakage of Intracellular Ions
The leakage of ions was assessed according to a previously published protocol [19] with minor modifications. Bacterial suspensions of C. acnes and S. epidermidis were prepared. Isolated colonies (5-7 colonies) of C. acnes and S. epidermidis were inoculated in RCM and BHI broth then incubated at 37 • C for 48 h and 24 h, respectively. Following incubation, cells were centrifugated at 10,000 rpm for 10 min and washed twice with PBS; the optical density was adjusted at 600 nm to 1. The bacterial cells were then treated with the MBC of the selected EO or DMSO (negative control) for 6 h and centrifuged. After centrifugation, the amounts of phosphate (PO 4− ) and sulphur (S 2− ) ions were measured using the ICP spectrometry technique [19] at the Faculty of Agriculture, Ain Shams University, Cairo, Egypt.

Development of the Selected EO Nanoemulsion
Nanoemulsion was prepared by a low-energy method [22] using 41.85% (w/w) of water, 2 MIC of the selected EO, tween 80 (27.5% v/v), and PEG 400 (27.5% v/v) as the surfactant and co-surfactant, respectively. The EO, tween 80, and PEG 400 were stirred at 800 rpm using a magnetic stirrer (Fisatom, Brazil) for 30 min. Water was then added dropwise, and the mixture was stirred at 800 rpm for 60 min. In the current study, we formulated thyme EO, clindamycin 1% (positive control) and blank formula (negative control) into nanoemulsions. The nanoemulsions were stored at room temperature (20 ± 2 • C) and evaluated after 1, 7, 21 and 30 days of preparation.

Characterization of EO Nanoemulsion Determination of the Particle Size and Polydispersity Index
The size and size distribution of thyme oil nanoemulsion were determined by the dynamic light scattering method [23] using a Malvern Mastersizer (DLS, Zetasizer Nano ZS, Malvern instruments, Malvern, UK). The polydispersity index (PDI) was measured using Malvern instruments to assess the particle size distribution. Three samples were used for size determination and the average values ± SD were calculated.

Nanoemulsion Morphology Using TEM
The morphology of the prepared thyme oil nanoemulsion was characterized using ouldn'tTEM at an acceleration voltage of 80 KeV (model JEM-1230, JEOL, Tokyo, Japan), where one drop of the diluted microemulsion was dried on the surface of a carbon-coated copper grid then stained with 2% phosphotungstic acid and allowed to dry at room temperature for 10 min for TEM inspection [24].
2.12. In Vivo Acne Animal Model for Assessment of EO Nanoemulsion Efficacy 2.12.1. Assessment of the Irritability of the EO Nanoemulsion A preliminary experiment was performed to examine the possible irritability of the prepared nanoemulsion. In this preliminary experiment, 10 µL of the prepared nanoemulsion was applied epicutaneously to the ears of a group of healthy uninfected mice (n = 5 BALB/c mice). Mice were observed over 5 days for any signs of inflammation [25].

Experimental Design
For the assessment of the selected EO nanoemulsion efficacy in vivo, 63 BALB/c male mice were used in three separate experiments (n = 21). In each experiment, mice were divided into three groups (n = 7 mice/group/experiment). Acne infection and inflammation were induced by injecting the right ear with 20 µL of 10 10 CFU/mL C. acnes according to a previously established protocol [26] using a Hamilton syringe 50 µL model 705 RN (Reno, NV, USA). The microcomedone formation was observed 24-48 h after injection. Daily changes in the ear thickness were recorded using an electronic digital micrometer caliper (0-25 mm/0.001 mm). The appearance of microcomedones and the increase in mice ear thickness by ≥10% were considered as indicators of acne induction [26]. For infected mice ears, 20 µL of the prepared nanoemulsion (test group) was applied epicutaneously, or 1% clindamycin (positive control), or blank nanoemulsion formula (negative control) for 3-5 days. Daily changes in mice ear thickness and the general health of the mice were recorded. At the end of the experiment, the mice were euthanized, and mice ears were excised for viable bacterial counts, an inflammatory markers assessment and histopathological assay (Olympus BX50 microscope, Tokyo, Japan) at a magnification power of 400×. Digital photographs of mice ears were taken.

Assessment of the Anti-Inflammatory Activity of the Selected EO Nanoemulsion
The percent of post-treatment epicutaneous inflammation was calculated for all the animal groups using the following formula: %inflammation = ear thickness infected ear − ear thickness uninfected ear ear thickness infected ear (1) The percentage inhibition of inflammation for all the animal groups was calculated as described previously [27] using the following formula: The NF-κB was measured in mice ear tissue homogenate using the Mouse Nuclear Factor KB (NFKB) ELISA Kit (MyBio Source, San Diego, CA, USA). The samples were prepared according to the kit manual and NF-κB concentration was determined and compared among different treatment groups.

Histopathological Examination of Ears after Treatment with EO Nanoemulsion
For the histopathological examination, whole excised mice ears in the 10% formalin solution were preserved before preparing the histological sections using the paraffin method technique [28]. All sections in ascending grades of ethanol were dehydrated, cleared in xylene then embedded in paraffin wax. Transverse sections (4-5 µm, thickness) were mounted on glass slides and stained with hematoxylin and eosin (H&E) stains. All sections were examined for the evaluation of inflammatory responses.

Assessment of the Antimicrobial Activity of EO Nanoemulsion
For an in vivo assessment of the antimicrobial activity of the prepared nanoemulsion, excised mice ears were homogenized in saline. The homogenates were then diluted using different dilutions of bacterial suspension, and 10 µL was cultured on RCM agar plates. C. acnes colonies were counted after 48 h of anaerobic incubation at 37 • C. Bacterial counts were expressed as CFU/mL.

Statistical Analysis
Data were plotted and analyzed using GraphPad Prism 9, GraphPad Software Inc. (GraphPad Prism version 9.0.0 (86) for OS X (GraphPad Software, San Diego, CA, USA, www.graphpad.com). Data were presented as mean ± standard deviation (SD) of at least three independent biological experiments. A one-way ANOVA test with Tukey's multiple comparison was used for the analysis of the results of agar disc-diffusion, reduction in NF-KB among different treatment groups and the antimicrobial activity between EO-and clindamycin-treated mice in the in vivo experiment. The unpaired t-test was performed to compare the results of the effect of EO on membrane integrity and the leakage of intracellular ions after treatment of bacteria with EO, whereas the two-way ANOVA test was applied to assess the statistical difference between EO and standard antibiotics in the loss of nucleic acid. The Mann-Whitney test was performed to compare the effect of the EO and clindamycin nanoemulsions in lowering the rate of inflammation in the animal model. Significance was considered at p-value ≤ 0.05.

Screening of the Antimicrobial Activity of the Tested EOs by Disc-Diffusion Method
Tea tree, clove and thyme EOs exhibited the largest inhibition zones among the screened EOs using the agar disc-diffusion method, while DMSO showed no zone of inhibition against both bacterial strains. The effect of tea tree, clove and thyme EOs at 25% and 50% (v/v) against C. acnes was not significantly different from the standard antibiotics. However, the three mentioned EOs were significantly more effective than the standard antibiotics at 25% and 50% (v/v) against S. epidermidis p-value ≤ 0.05 (Figure 1).

Determination of the Minimum Inhibition Concentration and the Minimum Bactericidal Concentration of the Screened EOs
The MIC of the selected EOs was determined as the lowest concentration of the oils, resulting in bacterial inhibition. The most potent EOs against C. acnes and S. epidermidis were tea tree and thyme, showing MIC values against C. acnes of 0.053 g/mL and 0.026 g/mL, respectively ( Table 2). Tea tree and thyme EOs also showed the lowest MIC values of 0.053 g/mL against S. epidermidis. MBC results showed that tea tree and thyme EOs exhibited killing of the bacteria at concentrations equal to their MIC against both bacteria  (Table 3). These two oils showed the lowest MBC values against C. acnes and S. epidermidis compared to other EOs.
Microorganisms 2022, 10, x FOR PEER REVIEW 9 of 24 inhibition against both bacterial strains. The effect of tea tree, clove and thyme EOs at 25% and 50% (v/v) against C. acnes was not significantly different from the standard antibiotics. However, the three mentioned EOs were significantly more effective than the standard antibiotics at 25% and 50% (v/v) against S. epidermidis p-value ≤ 0.05 (Figure 1).

Determination of Chemical Composition of the Most Effective EOs by Gas Chromatography-Mass Spectroscopy
The GC-MS analysis of tea tree, clove and thyme EO constitutes showed that the most abundant volatile compound of tea tree was 4-terpinenyl acetate (66.82%) (Supplementary Table S1

Determination of the Minimum Biofilm Inhibitory and Eradication Concentration of the Most Potent EOs
The selected EOs were tested for their anti-biofilm activity against S. epidermidis. Thyme EO showed the highest biofilm inhibition at half of its MIC of 0.053 g/mL. However, tea tree and clove EOs had anti-biofilm activity against biofilm formed by S. epidermidis at double their MIC of 0.107 g/mL and 0.274 g/mL, respectively (Table 5). Moreover, EOs were assayed for their anti-biofilm activity against S. epidermidis preformed biofilm. Thyme EO showed the highest biofilm eradication at its MIC of 0.107 g/mL, while clove EO had anti-biofilm activity against the biofilm formed by S. epidermidis at double of its MIC of 0.274 g/mL. However, tea tree oil was the least effective EO as it did not affect the preformed biofilm of S. epidermidis at a concentration of up to 0.215 mg/mL (4 its MIC) ( Table 5).

Determination of Time-Kill Kinetics of Selected EO
The antimicrobial testing results proves that thyme EO had the strongest antimicrobial activity and antibiofilm effect compared to the other tested EOs. Hence, thyme EO was selected for further analysis and characterization. Thyme EO had a bactericidal action after 10 h and 6 h of incubation against both C. acnes and S. epidermidis, respectively. The killing efficacy of thyme EO on S. epidermidis was higher than that of C. acnes at 6, 8 and 12 h (Figure 2).

Determination of the Minimum Biofilm Inhibitory and Eradication Concentration of the Most Potent EOs
The selected EOs were tested for their anti-biofilm activity against S. epidermidis. Thyme EO showed the highest biofilm inhibition at half of its MIC of 0.053 g/mL. However, tea tree and clove EOs had anti-biofilm activity against biofilm formed by S. epidermidis at double their MIC of 0.107 g/mL and 0.274 g/mL, respectively (Table 5). Moreover, EOs were assayed for their anti-biofilm activity against S. epidermidis preformed biofilm. Thyme EO showed the highest biofilm eradication at its MIC of 0.107 g/mL, while clove EO had anti-biofilm activity against the biofilm formed by S. epidermidis at double of its MIC of 0.274 g/mL. However, tea tree oil was the least effective EO as it did not affect the preformed biofilm of S. epidermidis at a concentration of up to 0.215 mg/mL (4 its MIC) ( Table 5).

Determination of Time-Kill Kinetics of Selected EO
The antimicrobial testing results proves that thyme EO had the strongest antimicrobial activity and antibiofilm effect compared to the other tested EOs. Hence, thyme EO was selected for further analysis and characterization. Thyme EO had a bactericidal action after 10 h and 6 h of incubation against both C. acnes and S. epidermidis, respectively. The killing efficacy of thyme EO on S. epidermidis was higher than that of C. acnes at 6, 8 and 12 h ( The mechanism of action of thyme EO on membrane permeability was shown in TEM photographs of C. acnes and S. epidermidis (Figure 3). Treatment of bacteria with thyme EO at its MBC led to bacterial cell wall damage and leakage of cytoplasmic content. TEM results suggested that thyme EO interferes with bacterial membrane integrity.

Visualization of the Effect of Thyme EO on Biofilm Formation by Scanning Electron Microscopy
The SEM was chosen for analyzing the surface and morphological changes in biofilm cells exposed to thyme EO. Regarding C. acnes and S. epidermidis biofilm formation, cellular adhesion and aggregation were detected in the untreated biofilm (control) (Supplementary Figure S4A,C); however, for biofilm treated with thyme EO, a lower number of adherent cells were observed (Supplementary Figure S4B,D).

Effect of Thyme EO on Bacterial Membrane Integrity Effect of Thyme EO on Potassium Ion (K + ) Permeability
The effect of thyme EO on the membrane integrity of bacterial cells was assessed through the leakage of potassium ions (K + ), as shown in Figure 4. Treatment of C. acnes and S. epidermidis with thyme EO at its MBC for 6 h has led to an observed leakage of K + . The mechanism of action of thyme EO on membrane permeability was shown in TEM photographs of C. acnes and S. epidermidis (Figure 3). Treatment of bacteria with thyme EO at its MBC led to bacterial cell wall damage and leakage of cytoplasmic content. TEM results suggested that thyme EO interferes with bacterial membrane integrity.

Effect of Thyme EO on Bacterial Membrane Integrity
Effect of Thyme EO on Potassium Ion (K + ) Permeability The effect of thyme EO on the membrane integrity of bacterial cells was assessed through the leakage of potassium ions (K + ), as shown in Figure 4. Treatment of C. acnes and S. epidermidis with thyme EO at its MBC for 6 h has led to an observed leakage of K + .

Effect of Thyme EO on 260 nm Absorbing Material (Nucleic Acids)
The OD 260nm of filtrates from control suspensions were not significantly different after 30, 60 and 120 min. Significant increases in the OD 260nm occurred after 30 min of treatment with thyme EO ( Figure 5). The OD 260nm of filtrates from control suspensions were not significantly different after 30, 60 and 120 min. Significant increases in the OD 260nm occurred after 30 min of treatment with thyme EO (Figure 5).

Effect of Thyme EO on the Leakage of Intracellular Ions
The effect of adding thyme EO at its MIC on the phosphorus (PO 4−) and sulfur (S 2− ) leakage in both C. acnes and S. epidermidis is shown in Figure 6. Compared to the DMSO control, leakage was observed after EO addition. These results suggest that increased membrane permeability is a factor in the mechanism of antimicrobial action.

Determination of the Particle Size and Polydispersity Index (PDI)
The z-average diameter recorded for the prepared microemulsion was 77.32 nm with a PDI of 0.38. This small particle size, together with its narrow distribution, provides high microemulsion stability, indicates the presence of droplet microemulsion and explains the clarity and isotropicity of the formulation, as shown in Supplementary Figure S5.

Nanoemulsion Particles Morphology Using TEM
The TEM micrograph of the prepared thyme oil nanoemulsion is illustrated in Supplementary Figure S6. Dark droplets were observed, almost spherical in shape, with the possibility to notice the outermost layer. Dark droplets were observed probably due to the strong interaction between the oil and the phosphotungstic acid used in staining the droplets.  A two-way ANOVA test was performed, p-value < 0.0001. ns means not statistically significant.

In Vivo Acne Animal Model for Assessment of EO Nanoemulsion Efficacy
Essential oil dose in the formulation was adjusted to 2 MICs (0.052 mg/mL) to avoid possible irritation or hypersensitivity on skin. The anti-acne activity of the formulated nanoemulsion of thyme EO was assessed using an in vivo acne mouse model. BALB/c right ears of mice were injected intradermally with C. acnes. After two days, the thyme nanoemulsion, 1% clindamycin or blank formulae was applied epicutaneously on the left ears of mice, and anti-inflammatory and antimicrobial activity against C. acnes were assessed (Figure 7).

Assessment of the Irritability of the Thyme Nanoemulsion
To avoid any possible hypersensitivity skin reaction, thyme nanoemulsion was applied to mice ear tissue before initiating the in vivo experiment. Mice were observed over five days for any sign of inflammation. Animals showed no inflammation or irritability when the thyme nanoemulsion formula was applied to healthy uninfected mice ears. The effect of adding thyme EO at its MIC on the phosphorus (PO 4−) and sulfur (S 2− ) leakage in both C. acnes and S. epidermidis is shown in Figure 6. Compared to the DMSO control, leakage was observed after EO addition. These results suggest that increased membrane permeability is a factor in the mechanism of antimicrobial action.

Determination of the Particle Size and Polydispersity Index (PDI)
The z-average diameter recorded for the prepared microemulsion was 77.32 nm with a PDI of 0.38. This small particle size, together with its narrow distribution, provides high microemulsion stability, indicates the presence of droplet microemulsion and explains the clarity and isotropicity of the formulation, as shown in Supplementary Figure S5

Nanoemulsion Particles Morphology Using TEM
The TEM micrograph of the prepared thyme oil nanoemulsion is illustrated in Supplementary Figure S6. Dark droplets were observed, almost spherical in shape, with the possibility to notice the outermost layer. Dark droplets were observed probably due to the strong interaction between the oil and the phosphotungstic acid used in staining the droplets.

In Vivo Acne Animal Model for Assessment of EO Nanoemulsion Efficacy
Essential oil dose in the formulation was adjusted to 2 MICs (0.052 mg/mL) to avoid possible irritation or hypersensitivity on skin. The anti-acne activity of the formulated nanoemulsion of thyme EO was assessed using an in vivo acne mouse model. BALB∕c right ears of mice were injected intradermally with C. acnes. After two days, the thyme nanoemulsion, 1% clindamycin or blank formulae was applied epicutaneously on the left Figure 6. Extracellular concentration of phosphate (PO 4− ) and sulfur ions (S 2− ) in aliquots of C. acnes (A) and S. epidermidis (B) after treatment with DMSO (control) and thyme EO at its MBC for 6 h. Data are represented as mean ± SD of three independent experiments. A multiple unpaired t-test test was performed, p-value < 0.001.

Morphological and Histopathological Assessment of the Healing Activity of the Nanoemulsion
Photographic pictures of BALB/c mice ear skins at the end of the experiment showed gross healing in mice treated with the thyme nanoemulsion. Moreover, the histopathological analysis of mice ears showed that thyme nanoemulsion was a potent anti-acne agent, as mice ears were completely healed. No necrotic dermatitis was detected, and normal dermatitis was observed after the thyme nanoemulsion was applied (Figure 8 and Table 6).

Assessment of Anti-Inflammatory Activity of the Thyme Nanoemulsion
The percentage of inflammation inhibition was calculated for all animal groups. The rate of reduction in mice ear thickness post-treatment with thyme EO was superior to the clindamycin-positive control (Figure 9). Moreover, inhibition of inflammation at the end of the experiment was significantly higher in mice treated with thyme nanoemulsion compared to the clindamycin-positive control (Figure 9).

Assessment of the Irritability of the Thyme Nanoemulsion
To avoid any possible hypersensitivity skin reaction, thyme nanoemulsion was applied to mice ear tissue before initiating the in vivo experiment. Mice were observed over five days for any sign of inflammation. Animals showed no inflammation or irritability when the thyme nanoemulsion formula was applied to healthy uninfected mice ears.

Morphological and Histopathological Assessment of the Healing Activity of the Nanoemulsion
Photographic pictures of BALB/c mice ear skins at the end of the experiment showed gross healing in mice treated with the thyme nanoemulsion. Moreover, the histopathological analysis of mice ears showed that thyme nanoemulsion was a potent anti-acne agent, as mice ears were completely healed. No necrotic dermatitis was detected, and normal dermatitis was observed after the thyme nanoemulsion was applied ( Figure  8 - Table 6). 3.8.4. Assessment of Anti-Inflammatory Activity of the Thyme Nanoemulsion by Measuring NF-κB Levels NF-κB concentration in mice ear tissue was determined using the ELISA kit. The result showed no significant difference between the thyme-treated mice group and the healthy uninfected mice group. Moreover, thyme nanoemulsion resulted in a significant 5-fold reduction in NF-κB level, whereas clindamycin nanoemulsion led to only a 2.5-fold reduction in NF-κB. This result confirms the anti-inflammatory effect of the EO (Figure 9).

Assessment of the Antibacterial Activity of the Prepared Nanoemulsion
The in vivo antimicrobial activity of thyme EO nanoemulsion against C. acnes was tested. Bacterial load was expressed as Log10 CFU/mL. There was no significant difference between thyme EO and clindamycin nanoemulsions regarding the antimicrobial activity, as both resulted in a reduction in the bacterial count compared to the blank formula ( Figure 9).

Nanoemulsion
Photographic pictures of BALB/c mice ear skins at the end of the experiment showed gross healing in mice treated with the thyme nanoemulsion. Moreover, the histopathological analysis of mice ears showed that thyme nanoemulsion was a potent anti-acne agent, as mice ears were completely healed. No necrotic dermatitis was detected, and normal dermatitis was observed after the thyme nanoemulsion was applied ( Figure  8 - Table 6).   In vivo antimicrobial activity of thyme EO nanoemulsion and clindamycin control exerting potent antimicrobial activity against C. acnes as the bacterial load was reduced to ~10 3 cfu/mL in both groups, while the bacterial load in the blank formula-treated mice group (negative control) was kept at 10 5 cfu/mL. Ordinary one-way ANOVA-Tukey's multiple comparison test was performed, pvalue < 0.0001. Data are represented as mean ± SD of three independent experiments.

Discussion
The microbial loads of C. acnes and S. epidermidis were found to increase simultaneously in acne vulgaris, which indicates their important role in the development and regulation of acne disease [29,30]. Therefore, C. acnes and S. epidermidis were chosen to be the targets for the assessment of the anti-acne natural drugs. The mainstay treatment of acne vulgaris involves the use of antibiotics such as erythromycin and clindamycin through their effects on acne-causing microbes [31][32][33]. However, the overuse of antibiotics in acne treatment is associated with the risk of emerging antibiotic resistance. Consequently, to overcome antibiotic resistance and to minimize the high cost of treatment, plants have been studied as an alternative therapy for acne.
In the search for alternative medicines, researchers sought natural products such as EOs, which have been used extensively as pharmaceutical agents for the management of various diseases including acne vulgaris with no reported antimicrobial resistance. EOs are secondary metabolites produced by many aromatic plants that contain a mixture of numerous types of bioactive molecules [34]. EOs have potent antimicrobial properties against a wide spectrum of pathogens including Gram-positive and Gram-negative bacteria. In a previous study, several EOs, including eucalyptus, tea tree, thyme white, lavender, lemon, lemongrass, cinnamon, grapefruit and clove buds were tested against the Staphylococcus aureus, Streptococci and Candida strain, and the EO of thyme white, lemon, lemongrass and cinnamon oil demonstrated that they were effective against these problematic bacteria [35].
In this study, we investigated the antimicrobial and anti-inflammatory activity of five of the most commonly used EOs belonging to the family Lamiaceae and Myrtacaea, and their mechanisms of action as anti-acne agents. Tea tree EO is currently used commercially in antiacne OTC products [36] with known anti-acne activity. Three of the selected EOs-thyme, basil and mentha-belong to the Lamiaceae family, known for having members with antimicrobial activity.
Thyme, tea tree and clove EOs inhibited the growth of C. acnes and S. epidermidis when tested by the disc-diffusion method. These results are in accordance with the study by [33], where Oregano vulgare and Thymus vulgaris oils showed bacteriostatic effects against Grampositive and Gram-negative bacterial strains. It was also proven that Oregano vulgare and Thymus vulgaris oils were more potent than Ocimum basilicum oil, because the latter contains estragole, which lacks the antibacterial properties of thymol and carvacrol contained in the former oils [37].
The bactericidal activities of thyme, clove and tea tree EOs were confirmed by the MBC values similar to their corresponding MICs (Tables 2 and 3) as well as the time-kill kinetics curves (Figure 2). The strongest and fastest bactericidal effect was shown by thyme EO, causing a total elimination of the initial bacterial inoculum after 10 and 6 h of exposure against C. acnes and S. epidermidis, respectively. In a previous study, the in vitro anti-acne potentials of tea tree, eucalyptus and thyme oils were also validated against C. acnes and S. epidermidis as antibacterial agents [38].
In the pathogenesis of acne, S. epidermidis plays a role in biofilm formation. We, therefore, assessed the anti-biofilm activity of the most potent EOs. Our results show that thyme EO possessed the strongest anti-biofilm activity.
The high potency of thyme EO can be attributed to its lipophilic property and its constituents which target the bacterial membranes [39]. The analysis by GC-MS of thyme EOs showed that thymol was its principal phenolic component (>70%) (Table 4).
Several studies have attempted to elucidate the modes of action of EOs; however, the antibacterial mechanisms are still not clear. Some EO constituents have been shown to penetrate the peptidoglycan layer and act on the cytoplasmic membrane of bacteria. As a result, a leakage of bacterial cell contents occurs [39]. In this study, the possible mechanisms of action of the most potent EO, thyme EO, was investigated against acnecausing bacteria. It was concluded that thyme EO had a bactericidal and anti-biofilm activity against C. acnes and S. epidermidis. As demonstrated, thyme EO exerted its antimicrobial activity by affecting the cell membrane of acne-associated bacteria where a leakage of cytoplasmic components was observed. It was evidenced that thyme EO caused the loss of nucleic acids, increased potassium permeability and leakage of intracellular components (Figures 4 and 5). The mechanism of action of the selected EO results was summarized in Figure 10. The SEM results confirmed the anti-biofilm effect of thyme EO on both C. acnes and S. epidermidis biofilms. In fact, Ref. [40] showed that thymol-rich oregano EO caused injury to Escherichia coli and Bacillus Subtilis by disrupting the cell membrane where a lack of cytoplasm was observed.
Several studies have attempted to elucidate the modes of action of EOs; however, the antibacterial mechanisms are still not clear. Some EO constituents have been shown to penetrate the peptidoglycan layer and act on the cytoplasmic membrane of bacteria. As a result, a leakage of bacterial cell contents occurs [39]. In this study, the possible mechanisms of action of the most potent EO, thyme EO, was investigated against acnecausing bacteria. It was concluded that thyme EO had a bactericidal and anti-biofilm activity against C. acnes and S. epidermidis. As demonstrated, thyme EO exerted its antimicrobial activity by affecting the cell membrane of acne-associated bacteria where a leakage of cytoplasmic components was observed. It was evidenced that thyme EO caused the loss of nucleic acids, increased potassium permeability and leakage of intracellular components (Figures 4 and 5). The mechanism of action of the selected EO results was summarized in Figure 10. The SEM results confirmed the anti-biofilm effect of thyme EO on both C. acnes and S. epidermidis biofilms. In fact, [40] showed that thymol-rich oregano EO caused injury to Escherichia coli and Bacillus Subtilis by disrupting the cell membrane where a lack of cytoplasm was observed. The in vitro studies corroborated that thyme EO was the most potent antimicrobial; therefore, it was prepared as a nanoemulsion formula to be assessed as an anti-acne agent in vivo. Nanoemulsions offer a solution for the low solubility of EO and enhance its application into the skin. In the current study, thyme EO at its double MIC (0.053 mg/mL) and 1% clindamycin (standard antibiotic) were formulated into nanoemulsions. The lowenergy method was used to prepare the nanoemulsion [22] and the formulae were used to assess thyme EO in vivo using an acne animal ear mouse model.
To assess the efficacy of anti-acne agents, the suppression of inflammation and decease in bacterial load along with the analysis of histopathological tissues are evaluated in acne animal models [25,27,41,42]. The healing effect of thyme EO was demonstrated through the reduction in comedonal lesions, the reduction in ear thickness, the histological The in vitro studies corroborated that thyme EO was the most potent antimicrobial; therefore, it was prepared as a nanoemulsion formula to be assessed as an anti-acne agent in vivo. Nanoemulsions offer a solution for the low solubility of EO and enhance its application into the skin. In the current study, thyme EO at its double MIC (0.053 mg/mL) and 1% clindamycin (standard antibiotic) were formulated into nanoemulsions. The lowenergy method was used to prepare the nanoemulsion [22] and the formulae were used to assess thyme EO in vivo using an acne animal ear mouse model.
To assess the efficacy of anti-acne agents, the suppression of inflammation and decease in bacterial load along with the analysis of histopathological tissues are evaluated in acne animal models [25,27,41,42]. The healing effect of thyme EO was demonstrated through the reduction in comedonal lesions, the reduction in ear thickness, the histological examination and the reduction in NF-KB and inflammatory mediators in thyme EO-treated mice ear tissue ( Figure 9). Additionally, thyme EO nanoemulsion significantly lowered the C. acnes bacterial load. The in vivo mice model results confirmed the antimicrobial and anti-inflammatory activities of thyme EO against C. acnes. To the best of our knowledge, this is the first study to report thyme EO nanoemulsion as a possible alternative anti-acne therapeutic agent.

Conclusions
Microbiological screening showed that thyme, tea tree and clove EOs inhibited the growth of C. acnes and S. epidermidis. The strongest and fastest bactericidal effect was recorded with thyme EO, where a total elimination of the initial bacterial inoculum after 10 h and 6 h of exposure against C. acnes and S. epidermidis, respectively, was observed. In addition, thyme EO exhibited the greatest anti-biofilm activity. The antimicrobial activity of thyme EO was exerted by affecting the cell membrane of acne-associated bacteria causing the leakage of its cytoplasmic components. The TEM results showed morphological alterations of C. acnes and S. epidermidis post-treatment with the MBC of thyme EO. The GC-MS analysis of thyme EO proved that thymol was the main phenolic compound of the oil. In this study, thyme EO was found to be the most potent antimicrobial EO and was formulated as a nanoemulsion topical dosage form. When tested in an acne-induced BALB/c model, this nanoemulsion had healing effects through the reduction in comedonal lesions, the reduction in ear thickness, and the reduction in NF-KB and inflammatory mediators when compared to clindamycin. Additionally, the thyme nanoemulsion significantly lowered the C. acnes bacterial load. In conclusion, the in vivo mice model results confirmed the antimicrobial and anti-inflammatory activities of thyme EO against C. acnes.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms10091874/s1, Figure S1: GC-MS chromatogram of tea tree EO; Figure S2: GC-MS chromatogram of clove EO; Figure S3: GC-MS chromatogram of thyme EO; Figure S4: Scanning electron microscopy (SEM) of thyme EO antibiofilm activity; Figure S5: Size distribution by intensity of the developed thyme oil nanoemulsion; Figure S6: TEM image of the developed thyme oil nanoemulsion, confirming that the formulation size is nanoparticle; Table S1: GC-MS analysis of volatile compounds in tea tree EO; Table S2

Data Availability Statement:
The raw data generated during the current study are available from the corresponding author upon request.